There is a need to develop renewable biofuels and chemicals that will help meet global demands for energy and synthetic chemistry feedstock, but without contributing to climate change or other environmental degradation.
Terpenoids represent the largest and most diverse group of naturally occurring organic compounds, and are all derived from the monomeric isoprene five-carbon building block. More than 25,000 different naturally occurring terpenoids have been identified, and many have plant origin. Terpenoids are classified into groups based on the number of five-carbon isoprene units they comprise; monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (greater than C40). β-Phellandrene (C10H16) offers example of such monoterpenes as a constituent of the essential oils synthesized by many plant species. It has significant commercial potential for use in the cosmetics and personal care industries, in cleaning products for household and industrial use, and medicinal use. There is also potential for β-phellandrene and other monoterpenes to be developed as feedstock in the synthetic chemistry and pharmaceutical industries, and as a renewable biofuel, where β-phellandrene itself may serve as supplement to gasoline or oligomerization of such monoterpene units may generate second order fuel molecules, suitable for use as supplements to jet fuel and diesel.
A number of plant species naturally produce β-phellandrene as a constituent of their essential oils, including lavender and grand fir. Essential oils are produced and stored in specialized organs called glandular trichomes, which form on the surface of leaves and flowers. Essential oils are mainly composed of monoterpenes and function in chemical defense against potential herbivores. The harvesting of essential oils from glandular trichomes, and subsequent purification of individual monoterpenes, such as β-phellandrene, is labour intensive and costly with relatively limited yields. The use of microorganisms, both photosynthetic and non-photosynthetic, for the production of such commercially useful and valuable chemicals is an attractive alternative to harvesting the product from plants.
All terpenoids are produced by two biosynthetic pathways: 1) the mevalonic acid (MVA) pathway, which operates in the cytosol of eukaryotes and archaea; and 2) the methyl-erythritol-4-phosphate (MEP) pathway, which is of prokaryotic bacterial origin and present in cyanobacteria, as well as in plant and algal plastids (see, FIG. 1). Synthesis of β-phellandrene in plants is due to the presence of a β-phellandrene synthase (β-PHLS) gene. This is a nuclear gene encoding a chloroplast-localized protein that catalyzes the conversion of geranyl diphosphate (GPP) to β-phellandrene. Plant β-phellandrene synthases, encoded by the gene β-PHLS, have been cloned and characterized from lavender, grand fir, tomato, and spruce (see, e.g., Demissie et al., Planta, 233:685-696 (2011); Bohlmann et al., Arch. Biochem. Biophys., 368:232-243 (1994); Schilmiller et al., Proc. Nat. Acad. Sci. U.S.A., 106:10865-10870 (2009); and Keeling et al., BMC Plant Biol. 11:43-57 (2011)).
Although photosynthetic microorganisms, such as microalgae and cyanobacteria utilize the MEP pathway, which generates GPP precursors, these microorganisms do not natively possess a β-phellandrene synthase gene or enzyme and thus, do not natively catalyze the conversion of GPP to β-phellandrene. However, they do express the MEP pathway and utilize the corresponding isoprenoid pathway enzymes for the biosynthesis of a great variety of needed terpenoid-type molecules like carotenoids, tocopherols, phytol, sterols, hormones, among many others) (see, FIG. 1). The MEP isoprenoid biosynthetic pathway (Lindberg et al., Metab Eng., 12:70-79 (2010)) consumes pyruvate and glyceraldehyde-3-phosphate (G3P) as substrates, which are combined to form deoxyxylulose-5-phosphate (DXP), as first described for Escherichia coli (Rohmer et al., Biochem. 1, 295:517-524 (1993)). DXP is then converted into methyl-erythitol phosphate (MEP), which is subsequently modified to form hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP). HMBPP is the substrate required for the formation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are terpenoid precursors. Cyanobacteria also contain an IPP isomerase that catalyzes the inter-conversion of IPP and DMAPP. In addition to reactants G3P and pyruvate, the MEP pathway consumes reducing equivalents and cellular energy in the form of NADPH, reduced ferredoxin, CTP and ATP, ultimately derived from photosynthesis. For reviews, see also (Ershov et al., J. Bacteriol. 184(18):5045-51; Sharkey et al., Ann. Bot. 101(1):5-18 (2002)).
Evidence in the literature shows that 15-carbon hydrophobic terpenoid hydrocarbons can be transgenically expressed in photosynthetic and fermentative microorganisms, but are trapped within the cell, where they are synthesized, requiring dewatering of the culture, drying of the biomass, followed by product extraction from within the cells. For example, the sesquiterpene β-caryophyllene was produced in a transgenic strain of the cyanobacterium Synechocystis. However, isolation of the product required an extensive protocol that included treating the isolated cellular biomass with an application of a chloroform:methanol:water solvent mixture to solubilize lipid bilayers, releasing all intracellular compounds, and extracting the lipophilic components (Reinsvold et al., J. Plant Physiol., 168: 848-852 (2011)).
Ten-carbon monoterpene hydrocarbon products occur in different distinct configurations, such as acyclic (e.g., myrcene), monocyclic (e.g., limonene and β-phellandrene), and bicyclic molecules (e.g., pinene). Spontaneous emission of monoterpene hydrocarbons from single-celled microorganisms to the extracellular space depends on the chemical nature of the monoterpene, and also depends on the lipid bilayer configuration and cell wall hydrophobic barriers imposed by the microorganism. For example, yield of limonene production increased substantially in transgenic E. coli upon the additional heterologous expression of an efflux pump from Alcanivorax borkumensis (AcrB/AcrD/AcrFa gene product; GenBank Accession No. YP692684) in the cell, suggesting limonene product feedback inhibition and/or toxicity to the cell.
The Lavandula angustifolia β-phellandrene synthase protein has been over-expressed in E. coli upon transformant cell induction with isopropyl β-D-1-thiogalactopyranoside, IPTG (Demissie et al., Planta, 233:685-696, 2011). However, IPTG induction in E. coli can be toxic to the cell, causing loss of cell fitness, thereby hindering a continuous and large scale production of β-phellandrene synthase by this method. Host cell toxicity could be due to accumulation of the recombinant protein itself and/or due to synthesis and intracellular accumulation of the transgenic product. The latter is one of the most common barriers in the commercial application of synthetic biology approaches for product generation.
This invention in based, in part, on the discovery of nucleic acids and expression systems that can be introduced and expressed in cyanobacteria and enable these microorganisms to produce β-phellandrene. Such genetically modified cyanobacteria can be used commercially in an enclosed mass culture system to provide a source of β-phellandrene which can be potentially developed as feedstock in the synthetic chemistry and pharmaceutical industries. For instance, β-phellandrene may serve as supplement to gasoline or oligomerization of such monoterpene units may generate second order fuel molecules, suitable for use as supplements to jet fuel and diesel.